The cellular mechanisms for biogenesis of (Na+K)-adenosine triphosphatase (Na,K-ATPase) have been investigated in the electric organ of the electric eel and in brine shrimp nauplii. In the electric organ, the relative incorporation retes into the large and small subunits of Na,K-ATPase were investigated. The ration of specific radioactivity in the small subunit to that in the large subunit was constant over various incorporation times and close to unity for incorporation in vitro and in vitro. Since the mole ration of the large and small subunits is probably one, the similar rates of incorporation indicate a coordinated synthesis, or assembly of the subunits into the holonenzyme, or both. For both subunits, there was a delay in incorporation of 2.5-3 h in vitro. This delay was demonstrated to be partially due to a slow equilbration of the amino acid precursor (0.5-1 h) and partially (1.5-2 h) due to post-translational processing. In brine shrimp nauplii, the biogenesis of Na,K-ATPase during initial development is being investigated. There are two large subunits of Na,K-ATPase during early development of the brine shrimp nauplii. The subunits are not artifacts of lipid oxidation or proteolysis during purification. They are both specifically phosphorylated from ATP in the presence of Na and Mg and dephosphorylated in the presence of K. Also copper phenanthroline corsslinking of Na, K-ATPase causes multimers of both bands of the large subunit without affecting any of the other protein bands. In the newly hatched nauplius, the two large subunits are about equal in concentration; whereas, later in development, the upper band appears as more prominent and the lower band is progressively lost. Preliminary results of radioactive incorporation into the brine shrimp Na,K-ATPase indicate that synthesis of Na,K-ATPase parallels that of other membrane proteins and occurs about 2-4 h in advance of enzyme activity. Further pulse-chase experiments are planned to determine if the lower band of the two large subunits is a precursor for the upper band or if the two bands are isozymes of Na,K-ATPase which occur at different times during development. Also we propose to study the biochemical differences between these two subunits by investigating their ouabain-binding site using photoactivatable derivatives of digitoxigenin.